A Study on Effect of Wetting on Mechanism of Coalescence in a Model Coalescer

In order to depict the mechanism of coalescence in fibrous bed coalescers, a model coalescer was fabricated. Both water/oil and oil/water dispersions were run through this model coalescer to check for coalescence on PTFE and glass surfaces. The equilibrium contact angle and the dynamic contact angle of the dispersed drops were measured on these surfaces in the presence of the continuous phase. Coalescence was monitored using a microscope. Based on these observations a mechanism of coalescence in the model coalescer is proposed. Different modes of coalescence are correlated to the equilibrium contact angle and the dynamic contact angle. Deposition of dirt on the coalescing surface is observed to result in change of wettability, leading to redispersion of the already coalesced dispersed phase into larger droplets.

Sydämen hypertrofian ja kroonisen vajaatoiminnan farmakologinen hoito

Sydämen krooninen vajaatoiminta on merkittävä maailmanlaajuinen ongelma. Se on erilaisten sydän- ja verisuonisairauksien aiheuttama monimuotoinen oireyhtymä. Sydämen vasemman kammion hypertrofia eli sydämen seinämien paksuuntuminen on yksi keskeinen tekijä, joka voi olla sydämen vajaatoiminnan taustalla. Kohonnut verenpaine on yleisin syy, joka johtaa sydänlihaksen paksuuntumiseen. Tämä johtaa sydämen pumppaustoiminnan häiriintymiseen, erilaisten neurohormonaalisten mekanismien aktivaatioon ja edelleen sydämen vajaatoimintaan. Sydämen vajaatoiminnan neurohormonaalisista mekanismeista tärkeimmät ovat reniini-angiotensiini-aldosteroni-järjestelmän ja sympaattisen hermoston aktivaatio, sydämen rakenteiden uudelleenmuovautuminen, sydänlihassolujen apoptoosi ja systeeminen tulehdustila. Sydämen hypertrofiaa ja sen syntymistä pyritään estämään kohonneen verenpaineen lääkehoidolla. Reniini-angiotensiini-aldosteronijärjestelmällä on keskeinen merkitys sydämen vajaatoiminnassa. Sydämen vajaatoiminnan ennusteeseen vaikuttavista lääkeaineista angiotensiinikonvertasin estäjät (ACEestäjät) ovat säilyttäneet johtoasemansa jo vuosikymmenten ajan. Angiotensiinireseptoreiden salpaajien (AT1-salpaajien) odotettiin syrjäyttävän ACE-estäjät sydämen vajaatoiminnan hoidossa, mutta toistaiseksi niitä pidetään vain vaihtoehtoisina lääkkeinä. Sympaattisen hermoston aktivaatiota vähentävät β-salpaajat ovat vakiinnuttaneet asemansa toiseksi tärkeimpänä lääkeryhmänä. Diureetit ovat paljon käytetty lääkeaineryhmä sydämen vajaatoiminnan hoidossa, mutta niistä ainoastaan aldosteroniantagonisteilla on tutkitusti ennustetta parantavaa vaikutusta. Kroonisen vajaatoiminnan hoidossa käytetään edelleen myös digoksiinia. Tulevaisuudessa sydämen vajaatoiminnan ennusteeseen vaikuttavia lääkeaineita voivat olla reniinin estäjät, neutraaliendopeptidaasin estäjät, vasopressiinin antagonistit tai inflammatroisiin sytokiineihin vaikuttavat molekyylit. Erikoistyön kokeellisessa osiossa tarkoituksena oli tutkia sydämen hypertrofian kehittymistä vatsa-aortta kuristetuilla rotilla ja kalsiumherkistäjä levosimendaanin sekä AT1-salpaaja valsartaanin vaikutuksia hypertrofian kehittymiseen. Kokeellisessa osiossa arvioitiin myös sydämen hypertrofian ja vajaatoiminnan jyrsijämallina käytetyn vatsa-aortan kuristuksen (koarktaation) toimivuutta ja vaikutuksia ultraäänen avulla määritettyihin kardiovaskulaarisiin parametreihin. Vatsa-aortta kuristettiin munuaisvaltimoiden yläpuolelta. Kuristus saa aikaan verenpaineen kohoamisen ja sydämen työtaakan lisääntymisen. Pitkittyessään tila johtaa sydänlihaksen hypertrofiaan ja vajaatoimintaan. 64 eläintä jaettiin ryhmiin, siten että jokaiseen ryhmään tuli kahdeksan eläintä. Ryhmistä kolmelle annettiin lääkeaineena levosimendaania kolmella eri päiväannoksella (0,01 mg/kg; 0,10 mg/kg; 1,00 mg/kg) ja kolmelle valsartaania kolmella eri päiväannoksella (0,10 mg/kg; 1,00 mg/kg; 10,00 mg/kg) juomaveden mukana. Lääkitys aloitettiin leikkauksen jälkeen ja jatkettiin kahdeksan viikon ajan. Kardiovaskulaariset parametrit, kuten isovolumetrinen relaksaatioaika (IVRT), vasemman kammion läpimitta systolessa ja diastolessa sekä seinämäpaksuudet, ejektiofraktio (EF), supistuvuusosuus (FS), minuuttitilavuus (CO) ja iskutilavuus (SV) määritettiin kahdeksan viikon kuluttua leikkauksesta ultraäänitutkimuksen avulla. Lisäksi määritettiin eläinten sydämen paino suhteessa ruumiin painoon. Tuloksia verrattiin ilman lääkehoitoa olleeseen koarktaatioryhmään. Eläinmallin toimivuutta arvioitiin vertaamalla koarktaatioryhmän tuloksia sham-operoidun ryhmän tuloksiin. Levosimendaanilla havaittiin työssä sydämen systolista toimintaa parantava vaikutus. Tämä näkyi tendenssinä parantaa ejektiofraktioita ja vasemman kammion supistuvuusosuuksia. Sydämen diastoliseen toimintaan ei kummallakaan lääkeaineella ollut merkittävää vaikutusta. Diastolista toimintaa arvioitiin isovolumetrisen relaksaatioajan muutoksilla. Sydämen hypertrofian kehittymiseen ei kummallakaan lääkeaineella ollut merkittävää vaikutusta. Eläinmallin todettiin mallintavan hyvin sydämen hypetrofiaa ihmisellä, mutta ei niinkään sydämen vajaatoimintaa.

Medetomidiinin glukuronidaation inhibitio

Lääkeainemetabolialla tarkoitetaan entsymaattisia reaktioita, jotka muuttavat lääkeaineita paremmin elimistöstä poistuvaan muotoon. Lääkeaineet voivat vaikuttaa toistensa metaboliaan inhiboimalla tai indusoimalla metaboloivia entsyymejä. Tällaisten interaktioiden seurauksena lääkeaineen pitoisuus elimistössä voi kasvaa jopa toksiseksi tai vähentyä merkittävästi. Tämä on erityisesti ongelmana silloin, kun käytössä on useita lääkkeitä samanaikaisesti. Lääketutkimuksessa onkin keskitytty tällaisten interaktioiden ennustamiseen ja niitä yritetään välttää tai ainakin vähentää. Työssä tutkittiin medetomidiinia, jonka on äskettäin havaittu metaboloituvan UDP-glukuronosyylitransferaasien (UGT) välityksellä. Työn tarkoituksena oli löytää medetomidiinin glukuronidaatiota inhiboivia yhdisteitä. Lisäksi haluttiin selvittää mahdollisen inhibition mekanismeja. On yleistä tutkia tietyn entsyymin substraatin interaktioita muiden saman perheen entsyymien kanssa. On kuitenkin harvinaisempaa tutkia tällaisia interaktioita kahden eri entsyymiperheen välillä. Tässä työssä tutkittiin inhiboivatko mahdolliset sytokromi P450 -entsyymiä (CYP) inhiboivat yhdisteet myös medetomidiinia glukuronoivia UDP-glukuronosyylitransferaaseja. Glukuronidaation inhibitiota tutkittiin HPLC-menetelmällä, joka on kehitetty aiemmin medetomidiinin glukuronidaation tutkimiseen. Aluksi glukuronidaatiota tutkittiin ilman inhibiittoreita. Tämän jälkeen tutkittiin kolmen mahdollisen inhibiittoriyhdisteen vaikutuksia medetomidiinin glukuronidaatioon ja tuloksia verrattiin ilman inhibiittoria saatuihin tuloksiin. Kolmen tutkitun yhdisteen havaittiin inhiboivan medetomidiinin glukuronidaatiota. Tutkimuksessa havaittiin myös mielenkiintoinen ilmiö, jossa inhibiittoriyhdisteen sitoutuminen aiheutti entsyymikineettisiä muutoksia UDP-glukuronosyylitransferaasin toiminnassa. On mielenkiintoista, että samat yhdisteet inhiboivat sekä CYP- että UGT-metaboliaa. Tulosten perusteella voidaan päätellä, että jos CYP ja UGT metaboloivat samaa yhdistettä, on mahdollista että yhdisteen rakenteelliset analogit aiheuttavat interaktioita molempien entsyymien kanssa. Uusia lääkeaineita kehitettäessä onkin otettava huomioon yleisesti tunnettujen CYP-entsyymien lisäksi myös UGT:t ja niiden mahdolliset yhteisvaikutukset.

Inverse relationship of the dehydrogenase and ADP-ribosylation activities in sodium-nitroprusside-treated glyceraldehyde-3-phosphate dehydrogenase is coincidental

Incubation of glyceraldehyde-3-phosphate dehydrogenase (GAPD) with sodium nitroprusside (SNP) decreased its activity in concentration- and time-dependent fashion in the presence of a thiol compounds, with DTT being more effective than GSH. Both forward and backward reactions were effected. Coinciding with this, HgCl2-sensitive labelling of the protein by [32P]NAD+ also increased, indicating the stimulation of ADP-ribosylation. Treatment with SNP of GAPD samples from rabbit muscle, sheep brain and yeast inactivated the dehydrogenase activity of the three, but only the mammalian proteins showed ADP-ribosylation activity. The SNP-modified protein of rabbit muscle GAPD, freed from the reagent by Sephadex filtration showed a concentration-dependent restoration of the dehydrogenase activity on preincubation with DTT and GSH. Such thiol-treated preparations also gave increased ADP-ribosylation activity with DTT, and to a lesser extent with GSH. The SNP-modified protein was unable to catalyze this activity with the native yeast enzyme and native and heat-inactivated muscle enzyme. It was possible to generate the ADP-ribosylation activity in muscle GAPD, by an NO-independent mechanism, on dialysis in Tris buffer under aerobic conditions , and on incubating with NADPH, but not NADH, in muscle and brain, but not yeast, enzymes. The results suggest that the inverse relationship of the dehydrogenase and ADP-ribosylation activities is coincidental but not correlated

Novel mechanism for high speed growth of transparent and conducting tin oxide thin films by spray pyrolysis

A novel mechanism is proposed for efficient manipulation of transport forces acting on the droplets during spray pyrolytic deposition of thin films. A ‘‘burst mode’’ technique of spraying is used to adjust the deposition conditions so as to transport the droplets under the new mechanism. Transparent, conducting thin films of undoped tin oxide prepared by this method showed significant improvement in growth rate. The films are found to be of fairly good quality with optical transmission of 82% and sheet resistance of 35 Ω/☒. The films are chemically homogeneous and grow preferentially along 〈200〉 direction.

Purification of 3,5-Dichlorocatechol 1,2-Dioxygenase, a Nonheme Iron Dioxygenase and a Key Enzyme in the Biodegradation of a Herbicide, 2,4-Dichlorophenoxyacetic acid (2,4-D), from Pseudomonas cepacia CSV90

An enzyme which cleaves the benzene ring of 3,5-dichiorocatechol has been purified to homogeneity from Pseudomonas cepacia CSV90, grown with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. The enzyme was a nonheme ferric dioxygenase and catalyzed the intradiol cleavage of all the examined catechol derivatives, 3,5-dichlorocatechol having the highest specificity constant of 7.3 μM−1 s−1 in an air-saturated buffer. No extradiol-cleaving activity was observed. Thus, the enzyme was designated as 3,5-dichlorocatechol 1,2-dioxygenase. The molecular weight of the native enzyme was ascertained to be 56,000 by light scattering method, while the Mr value of the enzyme denatured with 6 M guanidine-HCl or sodium dodecyl sulfate was 29,000 or 31,600, respectively, suggesting that the enzyme was a homodimer. The iron content was estimated to be 0.89 mol per mole of enzyme. The enzyme was deep red and exhibited a broad absorption spectrum with a maximum at around 425 nm, which was bleached by sodium dithionite, and shifted to 515 nm upon anaerobic 3,5-dichlorocatechol binding. The catalytic constant and the Km values for 3,5-dichlorocatechol and oxygen were 34.7 s−1 and 4.4 and 652 μM, respectively, at pH 8 and 25°C. Some heavy metal ions, chelating agents and sulfhydryl reagents inhibited the activity. The NH2-terminal sequence was determined up to 44 amino acid residues and compared with those of the other catechol dioxygenases previously reported.

Reaction of spironaphthalenones with hydroxylamine: Part I. A reinvestigation of the mechanism

Spironaphthalenones 1b–g on reaction with hydroxylamine hydrochloride gave the expected pyrrolotropones 2b–g. Furanotropone 6, postulated as an intermediate in the formation of pyrrolotropones, remained unchanged on reaction with hydroxylamine hydrochloride in ethanol. Reaction of unsymmetrical spironaphthalenones 1h–o with NH2OH.HCl gave the rearranged pyrrolotropones 2h–o.

Reaction of spironaphthalenones with hydroxylamine: Part III. A novel mechanism for the formation of products and trapping of an intermediate.

A mechanism involving the intermediacy of nitrene 5, formed from the oxime of spironaphthalenone 1 by acid catalysed dehydration, has been proposed to explain the formation of pyrrolotropones/pyrrolo esters from spironaphthalenones. The initially formed nitrene rearranges to the isopyrrole 6, which either undergoes sigmatropic migration to the pyrrolotropone 2 or adds alcohol to form the pyrrolo ester depending on substitution at 1′ position. The isopyrrole intermediate 6 has been trapped as a Diels-Alder adduct 8.

Identification of Critical Residues of the Mycobacterial Dephosphocoenzyme A Kinase by Site-Directed Mutagenesis

Dephosphocoenzyme A kinase performs the transfer of the c-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5-10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg2+ for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE.

Molecular diagnosis of developmental disorders

ABSTRACT Idiopathic developmental disorders (DDs) affect ~1% of the population worldwide. This being a considerable amount, efforts are being made to elucidate the disease mechanisms. One or several genetic factors cause 30-40% of DDs, and only 10% are caused by environmental factors. The remaining 50% of DD patients go undiagnosed, mostly due to a lack of diagnostic techniques. The cause in most undiagnosed cases is though to be a genetic factor or a combination of genetic and environmental factors. Despite the surge of new technologies entering the market, their implementation into diagnostic laboratories is hampered by costs, lack of information about the expected diagnostic yield, and the wide range of selection. This study evaluates new microarray methods in diagnosing idiopathic DDs, providing information about their added diagnostic value. Study I analysed 150 patients by array comparative genomic hybridization (array CGH, 44K and 244K), with a subsequent 18% diagnostic yield. These results are supported by other studies, indicating an enourmous added diagnostic value of array CGH, compared with conventional cytogenetic analysis. Nevertheless, 80% of the patients remained undiagnosed in Study I. In an effort to diagnose more patients, in Study IV the resolution was increased from 8.9 Kb of the 244K CGH array to 0.7 Kb, by using a single-nucleotide polymorphism (SNP) array. However, no additional pathogenic changes were detected in the 35 patients assessed, and thus, for diagnostic purposes, an array platform with ca 9 Kb resolution appears adequate. The recent vast increase in reports of detected aberrations and associated phenotypes has enabled characterization of several new syndromes first based on a common aberration and thereafter by delineation of common clinical characteristics. In Study II, a familial deletion at 9q22.2q22.32 with variable penetrance was described. Despite several reports of aberrations in the adjacent area at 9q associated with Gorlin syndrome, the patients in this family had a unique phenotype and did not present with the syndrome. In Study III, a familial duplication of chromosome 6p22.2 was described. The duplication caused increased expression of an important enzyme of the γ-aminobutyric acid (GABA) degradation pathway, causing oxidative stress of the brain, and thus, very likely, the mild mental retardation of these patients. These two case studies attempted to pinpoint candidate genes and to resolve the pathogenic mechanism causing the clinical characteristics of the patients. Presenting rare genetic and clinical findings to the international science and medical community enables interpretation of similar findings in other patients. The added value of molecular karyotyping in patients with idiopathic DD is evident. As a first line of testing, arrays with a median resolution of at least 9 Kb should be considered and further characterization of detected aberrations undertaken when possible. Diagnostic whole-exome sequencing may be the best option for patients who remain undiagnosed after high-resolution array analysis.

Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

HP0593 DNA-(N-6-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5'-GCAG-3' and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co2+, Mn2+, and also Mg2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.

Highly dispersed ultrafine Pt and PtRu nanoparticles on graphene: formation mechanism and electrocatalytic activity

We demonstrate a robust strategy for obtaining a high dispersion of ultrafine Pt and PtRu nanoparticles on graphene by exploiting the nucleation of a metal precursor phase on graphite oxide surfaces. Our method opens up new possibilities to engineer graphene-based hybrids for applications in multifunctional nanoscale devices.